ontrol of Cladosporium echinulatum in carnation using

bicarbonates and Trichoderma

Control de Cladosporium echinulatum en clavel mediante el uso
de bicarbonatos y Trichoderma.

Claudio Sandoval, Valeria Terreros, and Flavia Schiappacasse
Facultad de Ciencias Agrarias, Universidad de Talca, Casilla 747, Talca, Chile.
Corresponding author:fschiap@utalca.cl

Abstract
Carnations, Dianthus caryophyllus, have been cultivated in Chile for several decades as
a cut flower crop. At the present time, floriculture occupies 2,124 ha, of which 180 ha
are devoted to carnations. Almost all carnations produced are sold in the domestic
market. Several diseases affect carnations, among them fairyring leaf
spot(Cladosporium echinulatum), the most important foliar disease in Chile. Different
control alternatives for fairy-ring spot were evaluated on the carnation cv. Sarinah in
terms of disease incidence and severity. The study was performed in June-August 2003
with a commercial carnation planting in a greenhouse located in Champa(33 49' S,
70 45' W) that in previous growing seasons reported a high incidence of fairyring leaf
spot. The treatments included ammonium bicarbonate (5 g L
-1
), sodium bicarbonate (5
g L
-1
), ammonium bicarbonate + mineral oil Srpinguill (5 g L
-1
+ 10 mL L
-1
), sodium
bicarbonate + mineral oil Srpinguill (5 g L
-1
+ 10 mL L
-1
), mineral oil Srpinguill (10 mL
L
-1
), mancozeb (2 g L
-1
), copper oxichloride (2 g L
-1
), pentahydrated copper sulfate (1
mL L
-1
),Trichoderma virens strain Sherwood (Trichonativa) (5 mL L
-1
) and an untreated
control. Based on the results obtained, the most effective treatments were sodium
bicarbonate and T virens. Mancozeb, copper oxichloride, sodium bicarbonate + mineral
oil Springuill, ammonium bicarbonate, mineral oil Springuill and pentahydrated copper
sulfate were inefective in controlling carnation fairyring leaf spot.
Key words: Carnation, Cladosporium, Dianthus caryophyllus, foliar diseases, sodium
bicarbonate, Trichoderma.

Resumen
El clavel, Dianthus caryophyllus, ha sido cultivado en Chile para produccin de flores
de corte desde hace varas dcadas. Existen actualmente 2124 ha dedicadas a la
floricultura, de las cuales 180 ha son de clavel. Prcticamente toda la produccin de
esta especie se comercializa en el mercado interno. Numerosas son las enfermedades
que afectan su cultivo, siendo Cladosporium echinulatum el agente causal de la
mancha foliar (mancha anular) ms importante en esta especie. Se evaluaron distintas
alternativas de control de esta patologa en clavel cv. Sarinah, determinndose
incidencia y severidad de la enfermedad en las plantas. El estudio se realiz en junio-
agosto de 2003, en un plantel comercial bajo invernadero, ubicado en la localidad de
Champa(33 49' Latitud Sur, 70 45' Longitud Oeste), el cual tena antecedentes de
una alta incidencia de mancha anular. Los tratamientos fueron: bicarbonato de amonio
(5 g L
-1
), bicarbonato de sodio (5 gL
-1
), bicarbonato de amonio + aceite mineral
Springuill (5 gL
4
+ 10 mLL
-1
), bicarbonato de sodio + aceite mineral Springuill (5 gL
-1
+
10 mLL
-1
), aceite mineral Springuill (10 mLL
-1
), Mancozeb (2 gL
-1
), Oxicloruro de Cobre
(2 gL
-1
), Sulfato de cobre pentahidratado (Phyton-27) (1 mLL
-1
), Trichoderma
virens cepa Sherwood (Trichonativa) (5 mLL
-1
), y el testigo (sin aplicacin). Los
resultados indicaron que los tratamientos ms efectivos en el control de la enfermedad
fueron bicarbonato de sodio y Trichoderma virens cepa Sherwood, presentando las
mayores tasas de disminucin de incidencia (varas enfermas y hojas con lesin) y
severidad (lesiones activas por vara). Las plantas tratadas con mancozeb, oxicloruro
de cobre, bicarbonato de sodio + aceite mineral Springuill, bicarbonato de amonio,
aceite mineral Springuill y Sulfato de cobre pentahidratado presentaron los valores de
incidencia y severidad de la enfermedad ms altos, al final del experimento.
Palabras clave: Bicarbonato, Cladosporium, clavel, enfermedades
foliares, Trichoderma.

Introduction
According to the last agropecuarian census (ODEPA, 2007), there are 2,124 ha in Chile
dedicated to floriculture. Of this total, 180 ha are covered with carnations (Dianthus
caryophyllus LJ, which are primarily cultivated for internal consumption as cutflowers.
I
n
chile, four foliar diseases in carnations have been identified (Latorre, 2004), and
fairyring leaf spot, produced by Cladosporium echinulatum (Berk.) G.A. De Vries
(tel. Mycosphaerella dianthi (C.C. Burt) Jorst.), is the most important (Figure 1). This
pathogen causes a loss in quality and a reduction in the amount of harvested stems. It
affects the aerial organs of the plant, especially leaves and flowers. In leaves, the
manifestation begins with small purple spots, which limits commercialization (Cedeo
and Carrero, 1997). According to Agrios (1997) and Latorre ( 2004 )' conidia are
spread by wind, cultural practices, workers, diseased plants or inected harvest
residues.


There are multiple options for fungicides. For example, mancozeb is recommended as
a contact and preventive fungicide, and inhibits spore germination. Another option is
pentahydrate copper sulfate, a systemic product with healing and preventative actions.
This fungicide destroys the cell wall, inhibiting spore germination.
Ammonium bicarbonate, sodium bicarbonate and potassium carbonate have been
shown to be effective in the control of foliar pathogens, both in vitro and in vivo in
carnations and other ornamental species (Salazar, 2001; Palmer et al, 1997; Guzmn,
1994; Porter et al, 1993; Israel et al, 1993; Horst et al, 1992; Ziv and Zitter, 1992).
Fungi biocontrollers are considered a very ecological alternative, surviving for a long
time as dormant structures that may germinate again. Therefore, periodic
reinoculations are unnecessary (Wainwright, 1992). It has been proven that the
method used by the fungus Trichoderma spp. to control pathogenic fungi is primarily
through competition for food, space and predation (Cook and Baker, 1983).
Trichonativa (Bionativa, San Javier, Chile) is a liquid formulation based
on Trichoderma spp., with a concentration of 10
10
conidia mL
-1
plus hyphae pieces. It
supplies active mycelium, allowing for faster and more efficient action against
pathogens.
The objective of this study was to evaluate different alternatives to control carnation
fairyring leaf spot under greenhouse conditions based onthe incidence and severity
levels of the disease for the different treatments.
Materials and methods
Cultivation and place ofevaluation
The experiment took place in 2003 with a commercial carnation crop cv. Sarinah in a
greenhouse located in Champa (33 49'S, 70 45'W) that had previously been infected
by C. echinulatum.
Treatments
The treatments evaluated were as follows: (i) 5 g L
-1
of ammonium bicarbonate
(Droguera Mi-chelson, Santiago, Chile), (ii) 5 g L
-1
of sodium bicarbonate (Droguera
Michelson, Santiago, Chile), (iii) 5 g L
-1
of ammonium bicarbonate in combination with
10 mL L
-1
of mineral oil (formulated product, fe.) Springuill (ANASAC S. A., Santiago,
Chile), (iv) 5 g L
-1
of sodium bicarbonate in combination with 10 mL L
-1
of mineral oil
(fe.) Springuill, (v) 10 mL L
-1
of mineral oil (fe.) Springuill, (vi) 2 g L
-1
mancozeb
(Mancozeb 80% WP, Arysta Lifescience Chile S.A.), (vii) 2 gL
-1
of copper oxychloride
(Copper Oxychloride, Platte Chemical CO., U.A.P, Chile S.A.), (viii) 1 mL L
-1
of
pentahydrate copper sulfate (Phyton-27, Source Technology Biologicals, Inc, USA) and
(ix) 5 mL L
-1
ofTrichoderma virens strain Sherwood (Trichonativa, Bionativa, San
Javier, Chile). In addition, a control treatment without applications was included.
Application method
The application of the fungicides was carried out using a 5 L back sprayer (Solo,
Sindelfingen, Germany). The bicarbonates and the mineral oil alone and mixed were
applied every 7 days for a month, while treatments with mancozeb, copper oxychloride
and pentahydrate copper sulfate were applied every 10 days. T. virens strain
Sherwood was applied twice every 15 days.
In all cases, the wetting was performed with 0.5 L of water for each 0.7 m
2
plot up to
pouring point. Product drift was avoided by using a 1 x 1 m plstic barrier between
experimental units during application.
Evaluation
Incidence and severity evaluations were conducted every 7 days for 60 days (July-
August). Before the first application, a base evaluation of the state of each stem of the
experimental plot was carried out. Therefore, the number of infected stems by plot (I
1
)
and the number of leaves with C. echinulatum wounds per stem (I
2
) was determined.
These were estimated according to the following equation:

Calculation of the percent decrease in the incidence in stems and leaves was carried
out between the first and the fourth evaluations and between the fourth and the final
evaluations. This corresponds to the difference between the incidence values obtained
21 days after the start of the evaluations and 49 days after the start. In cases where
the incidence value of the last evaluation date was higher than that of the initial date,
a value of zero was assigned for the percent decrease.
Severity (S) was quantified according to the following equation (French and Hebert,
1980):

Wounds were considered active when the pres-ence of pathogen mycelium and conidia
was verified using a 10X manual magnifier.
Severity was measured each time incidence was evaluated. Estimations of decreases in
the severity of carnation ringspot were carried out using the same procedure described
above for I
1
and I
2
.
The rate of disease control was estimated based on the values of incidence or severity
obtained on the first evaluation day (1 DDIE). This value represented 100% infection.
Experimental design and statistical analysis
A completely randomized experimental design was used that considered four
repetitions and ten treatments. The experimental unit corresponded to two stems from
0.7 m
2
plots of carnation plants cv. Sarinah. The results obtained were submitted to an
analysis of variance. Comparisons of means were carried out using the Duncan test (p
0.05). The percent values of incidence (I
1
and I
2
) were transformed to angular
values (arcsen (%/100)
1/2
) before conducting statistical analysis.
Results
The use of T. virens had no effect on the percent decrease of the stems infected in the
first evaluation period. In the second period, sodium bicarbonate and T. virens stood
out, both differing statistically from the remaining treatments (Table 1). In the second
period of evaluation, T. virens with sodium bicarbonate treatment showed the greatest
effect in terms of decreasing the number of leaves with C. echinulatum wounds. On the
contrary, copper oxychloride, mancozeb, pentahydrate copper sulfate and ammonium
bicarbonate alone and with mineral oil did not differ statistically from the control
treatment (Table 1).


In decreasing the number of active wounds, within the first evaluation period (0 to 21
days post-application) copper oxychloride, sodium bicarbonate and ammonium
bicarbonate plus mineral oil were all statistically similar to one another. The remaining
treatments did not dif-fer statistically from the control (p 0.05). In the second
evaluation period, between days 21 to 49 post-application, T. virens was shown to be
the most effective treatment in terms of decreasing the number of active wounds. The
differences were shown to be highly statistically significant (p 0.01) with respect to
the rest of the treatments, which did not differ statistically from the control treatment
(Table 2).


In the first evaluation, carried out before the first applications of the products, there
were no significant differences between plots with regard to the number of stems
infected in relation to the total (I
1
) (Table 3). However, in the final evaluation, sodium
bicarbonate was found to be the most effective treatment in terms of the number of
diseased stems, and was statistically similar to treatment with T. virens. T.
virens treatment did not differ from the treatments with pentahydrate copper sulfate,
copper oxychloride and ammonium bicarbonate plus mineral oil. The carnation plants
treated with mancozeb, mineral oil, sodium bicarbonate plus oil and ammonium
bicarbonate presented the highest incidence values of the disease at the end of the
test, which were not statistically different from the control treatment.


At the beginning of the experiment, the average percentage of wounded leaves with
respect to the total for two stems (I
2
) did not present significant differences among
treatments, with all of the experimental plots showing similar percentages of carnation
leaves with active wounds. On the contrary, in the evaluations conducted at the end of
the test, sodium bicarbonate showedthe highest control rate for the disease (63%),
followed by T. virens38%). However, the control rate of I
2
was low. Six of the nine
evaluated products showed percentages lower than 25% (Table 4).


Sodium bicarbonate and T. virens presented control rates for disease severity of 74%
and 44%, respectively, expressed as the number of active wounds in leaves. The
treatments with mancozeb, pentahydrate copper sulfate and ammonium bicarbonate
had no effect on the control of C echinulatum active wounds, and were not different
from the control treatment (Table 5).


Discussion
All of the treatments had some effect on fairyring leaf spot control, differing
statistically from the incidence and severity obtained in the control treatment.
However, the most effective were sodium bicarbonate and T. virens.They presented
the highest decrease rates with respect to I
p
I
2
and severity (Tables 2, 3, and 4).
Although sodium bicarbonate acted effectively to control the pathogen in the first five
weeks of the test, from day 35 after the start of the applications, no effect was
observed with respect to a decrease in the number of active wounds. This indicates the
need for weekly application of the product; presumably, the disease can reach levels of
incidence and severity close to zero (Figure 2).


On the other hand, when comparing the effects of ammonium bicarbonate in
controlling C. echinulatum to its effects against other pathogens evaluated in other
tests, it seems quite ineffective. In celery (Apium graveolensvar. secalinum),
ammonium bicarbonate was the most effective product in septoria control (Septoria
apiicola) in vitro, exceeding sodium bicarbonate used at 1.0, 0.5 and 0.25%
concentrations (Salazar, 2001). Similar results were found by Ziv and Zitter (1992) in
the control of disease agents of horticultura! species such asSphaerotheca fuliginea,
Didymella bryoniae, Alternara cucumerina and Ulocladium cucurbitae when using
ammonium bicarbonate at 1% (p/v).
Ammonium bicarbonate plus mineral oil seems to be more effective than sodium
bicarbonate mixed with mineral oil. This was evident by the lack of healing activity of
sodium bicarbonate plus mineral oil. However, it is noteworthy that sodium
bicarbonate and mineral oil at 0.01% (v/v) alone or combined reduced the incidence of
carnation fairyring leaf spot, yielding a better effect when sodium bicarbonate was
applied separately. This conflicts with studies by Horst et al. (1992) on the control of
fungal diseases in ornamental plants with bicarbonates in Rosaspp. and Euonymus
japnica. Horst et al. (1992) made weekly applications of sodium bicarbonate at 0.063
M and Sunspray ultrafine mineral oil at 1% (v/v) and reduced the incidence of both
diseases, obtaining a better effect when applied together. The difference may be
explained by the fact that the mineral oil used in this experiment did not have the
ultra-refining features of the Sunspray mineral oil.
On the other hand, the bicarbonates with mineral oil used for the C.
echinulatum control created spots on carnation leaves.
Guzman (1994) and Salazar (2001) attributed the low healing activity showed by
bicarbonates to their lack of a systemic effect and low or nuil translocation capacity.
Accordingly to that, these products act only as preventive fungicides.
Regardless of the aforementioned study, Horst et al. (1992) and Ziv and Hagilady
(1993) indicate the healing actions of bicarbonates on powdery mildew in Rosa spp.
and Euonymus spp. These effects may be due to the corporeal structures of the fungus
causing the disease, which are more superficially exposed to the actions of these
products. In the present experiment, although all of the products were applied on both
faces of the carnation leaves, the only products that caused wound healing on the
upper sides and undersides of the leaves were the bicarbonatos and the
bioconfroller T. virens slrain Sherwood. Accordingly, bicarbonatos would have healing
effecls on C. echinulatum that could be exlrapolaled to other fungi with superficial
slruclures, such asPenicillium and Botrytis. The plant leaves where the other
treatments were applied presented active wounds on both faces and consanl
sporulalion, wilh masses of mycelia and conidia.
The mode of action of the bicarbonates in the control of phytopathogen fungi is still
unknown, as stated by Horstet al. (1992), Ziv and Zitter (1992), Ziv and Hagilady
(1993), Guzman (1994) and Salazar (2001). The fungicide action could be due to a
perturbation of the optimal pH for conidia germination and continued growth of the
germinal tube of the C. echinulatum fungus.
Guzman (1994) points out that the inhibition of germination caused by bicarbonates
in in vitro solutions could be associated with high levels of HC0
3
- or C0
3
= ions, which
could have fungicidal effects (Punja and Grogan, 1987).
On the other hand, the limited controlling effect of mancozeb is clear when the severity
levels at the beginning and end of the test are considered. The same is observed with
copper oxychloride and pentahydrate copper sulfate. However, copper oxychloride
reduced the wounds by 23.6%, making it more effective than mancozeb and
pentahydrate copper sulfate for disease control, perhaps due to the greater residual
effect of this product (Table 5).
According to the results, the biological controller was a good control alternative. An
important aspect to consider when including this controller as part of an integrated
pest management program is the mode of action, primarily competition. Once the
biocontroller is applied, Trichoderma sp. propagules develop abundantly, superficially
colonizing the plant tissues and competing for space with the fungus C. echinulatum.
In addition to this mode of action, mycoparasitism has been described, where certain
enzymes could be involved, such as -1,3 gluconase, lipase and proteolases, that are
liberated into the environment by Trichoderma sp. and destroy the structural
components of the pathogen (Elad et al, 1982).
With regard to the mycoparasitism, it is clear that the earlier the antagonist is applied
to the host, the better is the control achieved. Hong et al. (1998), Cook and Baker
(1989) indicate that the period between pathogen establishment and further
sporulation corresponds to the rnost vulnerable stage for the pathogen to be displaced
by the antagonist. In addition, by applying the inoculum of the biocontroller agent
before the pathogen propagules are formed or spread, more efficient control can be
achieved because the antagonist reaches a higher inoculum concentration when the
pathogen has not yet reached high sporulation. This could explain why the control of C.
echinulatum using T. virens strain Sherwood was efficient only from 28 days after the
applications began, showing a strong rate of severity decrease after that point (Figure
2). In fact, the daily decrease in disease severity from that time was equal to 0.8
active wounds per day. Therefore, it is of interest to plan a strategy for disease control
using the native strain Sherwood by considering preventive applications of the
biocontroller from a month before the appearance of the predisposing conditions for
the development of C. echinulatum. However, even under the conditions of this test, T.
virens strain Sherwood was able to control the damage caused by the pathogen,
presenting a healing effect on the active wounds of C. echinulatum on carnation
leaves.
These results confirm statements by Cook and Baker (1989) and Chet (1987), who
point out that Trichoderma is able to efficiently control a large number of diseases.
Figure 3 shows how the control treatment and the treatment with T. virens strain
Sherwood presented similar behaviors in the first month (0 a 28 days). However, the
control presented a sustained rate of increase inthe number of active wounds per stem
(0.34 wounds stem
-1
day
-1
).


It is noteworthy that the economic damage produced by C. echinulatum is high,
reaching close to 60% (data not presented) and restricting to a great extent the
production of carnation flowers due to its fast dissemination and aggressiveness under
greenhouse conditions. Therefore, the costs that arise when applying sodium
bicarbonate or T. virens Strain Sherwood (data not presented) are justified if the lower
profitability obtained with carnation cultivation infected with C. echinulatum is
considered.
Accordingly, when there are predisposing conditions for disease development inthe
months of June, July and August, applications of sodium bicarbonate and T.
virens strain Sherwood are an efficient control strategy to prevent infection by C.
echinulatum in carnations.
The best treatments to control carnation ring-spot, caused by the fungus C.
echinulatum, were sodium bicarbonate and T. virens strain Sherwood. Both presented
the lowest indexes of incidence and severity of the disease.
Sodium bicarbonate showed aneffective control of C. echinulatum until 35 days after
the start of the applications, which indicates a residual period of the product of seven
days.
The biological controller T. virens strain Sherwoood demonstrated effective control of
the pathogen from the fourth week after the applications were initiated. Therefore,
preventive applications must begin a month before the predisposing conditions for
fungus development occur.
Carnation plants treated with mancozeb, copper oxychloride, sodium bicarbonate plus
oil, ammonium bicarbonate, mineral oil and pentahydrate copper sulfate presented the
highest values of incidence and severity of the disease at the end of the experiment.

Referencias
AFIPA-IMPPA-SAG. 2002-2003. Manual Fitosanitario. Fundacin para el desarrollo
frutcola. Servicios de Impresin Lser. Santiago, Chile.1214 p. [ Links ]
Agrios, G. 2005. Plant Pathology (5th Edition). Elsevier Academic Press, San Diego,
California. USA. 948 pp. [ Links ]
Cedeo, L., and C. Carrero. 1997. Cladosporium echinulatum, causante de manchas en
hojas y flores del clavel en Mrida, Venezuela. Revista Forestal Venezolana 41:91-
92. [ Links ]
Chet, I. 1987. Innovative approaches to plant diseasecontrol. John Wiley and Sons,
NY, USA. 372 p. [ Links ]
Cook, R. J, and K.F. Baker. 1989. The nature and practice of biological control of plant
pathogens. American Phytopathological Society. St. Paul, MN, USA. 539
p. [ Links ]
Elad, Y, Y. Chet, and Y. Henis. 1982. Degradation of plant pathogenic fungi
by Trichoderma harzianum. Canadian Journal of Microbiology 28:719-
725. [ Links ]
French, E. R., y T.T. Hebert. 1980. Mtodos de investigacin fitopatolgica. Editorial
IICA, San Jos, Costa Rica. 289 p. [ Links ]
Guzmn, P 1994. Control de la roya del clavel a travs del uso de distintos tipos de
bicarbonatos y carbonato en mezcla con aceite mineral. Tesis de grado. Escuela de
Agronoma, Facultad de Ciencias, Agrarias Universidad de Talca. Talca, Chile. 84
p. [ Links ]
Hong, C, T. Michailides, and B. Holtz. 1998. Effecs of wounding, inoculum density, and
biological control agents on postharvest brown rot of stone fruits. Plant Disease
82:1210-1216. [ Links ]
Horst, R. K., S.O. Kawamoto, and L.L. Porter. 1992. Effect of sodium bicarbonate and
oils on the control of powdery mildew and black spot of roses. Plant Disease 76:247-
251. [ Links ]
Israel, H. W., S.W. Ingalls, L.L. Porter, and R.K. Horst. 1993. Control of powdery
mildew of rose with bicarbonates. A qualitative microscopic study of erradication.
Phytopathology 93:244 (Abstract). [ Links ]
Latorre, B. 2004. Enfermedades de las Plantas Cultivadas. Sexta ed. Ediciones
Universidad Catlica de Chile. Santiago, Chile. 638 p. [ Links ]
ODEPA. 2007. Flores de corte (en lnea). Chile. Avalaible online
at http://www.odepa.gob.cl. (Accessed: November, 2008). [ Links ]
Porter, L. L., R. K. Horst, and H.W. Israel. 1993. Control of powdery mildew of rose
with bicarbonates: II. A quantitative influence on fungal structures. Phytopathology
83:246 (Abstract). [ Links ]
Palmer, C. L., R.K. Horst, and R.W. Langhans. 1997. Use of Bicarbonates to inhibit in
vitro colony growth ofBotrytis cinerea. Plant Disease 81:1432-1438. [ Links ]
Salazar, A. 2001. Evaluacin de laActividad Fungicida de Dos Bicarbonatos y un
Carbonato en Condiciones in vitroe in vivo. Tesis presentada para obtener el grado de
Ingeniero Agrnomo. Escuela de Agronoma, Facultad de Ciencias Agrarias.
Universidad de Talca. Talca, Chile. 48 p. [ Links ]
Punja, Z.K., and R.G. Grogan. 1987. Effects of inorganic salts, carbonate-bicarbonate
anions, ammonia, and the modifying influence of pH on sclerotial germination
of Sclerotium rolfsii. Phytopathology 72:635-639. [ Links ]
Wingwright, M. 1992. An Introduction to Fungal Biotechnology. Wiley and Sons.
England. UK. 202 p. [ Links ]
Ziv, O., and A. Hagilady. 1993. Controlling powdery mildew in euonymus with polymer
coatings and bicarbonate solutions. HortScience 28:124-126. [ Links ]
Ziv, O., and T.A. Zitter. 1992. Effects of bicarbonates and film-forming polymers on
cucurbit foliar diseases. Plant Disease 76:513-517. [ Links ]

Received 14 November 2008. Accepted 02 April 2009.